Previously, we documented that activation of protein kinase C- (PKC-) mediates mitochondrial dysfunction in cultured renal proximal tubule cells (RPTC). This study tested whether deletion of the PKC- decreases dysfunction of renal cortical mitochondria and improves kidney function after renal ischemia. PKC- levels in mitochondria of ischemic kidneys increased 24h after ischemia. Complex I- and complex II-coupled state 3 respirations were reduced 44% and 27%, respectively, in wild-type (WT) but unchanged and increased in PKC--deficient (KO) mice after ischemia. Respiratory control ratio coupled to glutamate/malate oxidation decreased 50% in WT, but not in KO mice. Activities of complexes I, III, and IV were decreased 59, 89, and 61%, respectively, in WT but not in KO ischemic kidneys. Proteomics revealed increases in levels of ATP synthase (α-subunit), complexes I and III, cytochrome oxidase, α-ketoglutarate dehydrogenase, and thioredoxin-dependent peroxide reductase after ischemia in KO, but not in WT animals. PKC- deletion prevented ischemia-induced increases in oxidant production. Plasma creatinine levels increased 12-fold in WT and 3-fold in KO ischemic mice. PKC- deletion reduced tubular necrosis, brush border loss, and distal segment damage in ischemic kidneys. PKC- activation in hypoxic RPTC in primary culture exacerbated, whereas PKC- inhibition reduced decreases in: 1) complex I- and complex II-coupled state 3 respirations, and 2) activities of complexes I, III, and IV. We conclude that PKC- activation mediates: 1) dysfunction of complexes I and III of the respiratory chain, 2) oxidant production, 3) morphological damage to the kidney, and 4) decreases in renal functions after ischemia.